M-csfr Was Ligands Combined With The Cloning And Expression Of The Functional Areas

Total RNAs were extracted from human fresh placeta, and the cDNA fragment encoding for the M-CSFR extracellular region, the first three immunoglobulin(Ig)-like motifs(D1-3), which encodes the ligand-binding domains[Wang et al, Mol Cell Biol, 1993], was amplified by RT-PCR. The cDNA was then cloned into the expression vector pET28a downstream to the His-Tag site. After transformation into E.coli BL21( DE3) host bacteria, D1-3 was expressed efficiently and stably in inculsion bodies with the yield of 46% total bacteria proteins. The recombinant protein was purified by affinity chromatogaphy using a His Bind Resin column charged with nickel. The D1-3 showed a single band in SDS-PAGE and the apparent molecular weight was ~34 kD. Colony formation inhibition assay showed that D1-3 could obviously inhibit the colony formation of J6-1 leukemia cells at the final concentration of 1, 0.1 and 0.01 μM respectively, suggesting that the D1-3 possesses significantly biological activity. Anti-rabbit polyclonal antibodies against D1-...